Tissue transglutaminases (TGases) are GTP-binding/GTPases with an enzymatic (transamidation) activity that catalyzes the covalent linkage between glutamine residues and primary amino groups, resulting in the formation on new protein-protein and protein- polyamine complexes. The TGases have been implicated in a number of important biological responses including neuronal development and degeneration, as well as retinoic acid-induced cellular differentiation, and apoptosis. Both the GTP-binding and transamidation activities of the TGases are activated by retinoic acid. Recently, we identified two candidate target/binding partners for the TGase, namely, the eukaryotic initiation factor (eIF)5A, and the retinoblastoma gene product (Rb), both of which are essential for cell viability. We also have found that the TGase-catalyzed transamidation of Rb protects it against proteolytic degradation by caspases. The studies outlined in this application are aimed at understanding different aspects of the structure and function of this interesting dual function GTP- binding protein/transamidase and its role in retinoic acid- induced cellular differentiation. There are three specific aims. 1.) Delineate the mechanistic basis for the intramolecular coupling between the GTP-binding and transamidation activities of the TGase. We plan to determine how the TGase is able to bind and hydrolyze GTP and to establish how the GTP-binding/GTP hydrolytic cycle regulates the transamidation activity. The ultimate aim will be to generate TGase mutants that are defective in GTP-binding or that contain GTP-binding/GTP hydrolytic activity that is uncoupled from transamidation activity, since these mutants should be useful for cellular studies of TGase function. 2.) Determine how retinoic acid mediates the cellular regulation of the TGase. In these studies, we plan to determine whether a negative regulatory protein is responsible for maintaining the TGase in an inactive state in the absence or retinoic acid treatment, as well as begin to establish the molecular signals responsible for the retinoic acid-stimulated activation of the TGase. 3.) Establish a role for the interactions of the TGase with two new target/binding partners that are essential for cell growth. The main emphasis of these studies will be to determine whether the retinoic acid-dependent interactions of the TGase with Rb and eIF-5A are essential for ensuring cell viability when cells undergo cell-cycle arrest during differentiation. The results of these studies should yield new information regarding the regulation of an important dual function GTP-binding protein/transamidase and its role in mediating the balance between cellular differentiation and apoptosis.